Biosafety Considerations
for Research with
Lentiviral Vectors
December 2006
Biosafety Considerations for Research with Lentiviral
Vectors
Recombinant DNA Advisory Committee (RAC) Guidance Document
Background: The use of lentiviral vectors has been increasing because the vector
system has attractive features; however, such research also raises biosafety issues. The
NIH Office of Biotechnology Activities has received frequent questions regarding the
appropriate containment for lentiviral vectors, particularly those derived from HIV-1.
Because the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH
Guidelines) do not explicitly address containment for research with lentiviral vectors, the
RAC was asked to provide additional guidance for institutional biosafety committees
(IBCs) and investigators on how to conduct a risk assessment for lentiviral vector
research. At the March RAC 2006 meeting, the RAC offered the following findings and
recommendations.
Risks of lentivirus vectors: The major risks to be considered for research with HIV-1
based lentivirus vectors are:
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Potential for generation of replication-competent lentivirus (RCL), and
Potential for oncogenesis.
These risks can be mitigated by the nature of the vector system (and its safety features)
or exacerbated by the nature of the transgene insert encoded by the vector.
General criteria for risk assessment of lentivirus vectors: Decisions about
containment should take into account a range of parameters/considerations including:
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The nature of the vector system and the potential for regeneration of
replication competent virus from the vector components,
The nature of the transgene insert (e.g., known oncogenes or genes with high
oncogenic potential may merit special care)
The vector titer and the total amount of vector,
The inherent biological containment of the animal host, if relevant,
Negative RCL testing (see section below)
General containment considerations: Either BL2 containment or enhanced BL2
containment is often appropriate in the laboratory setting for research involving the use
of advanced lentivirus vector systems that have multiple safety features and that
segregate vector and packaging functions onto four or more plasmids. Enhanced BL2
containment may include in addition to attention to sharps (and use of safety needles
where feasible), the use of personal protective equipment intended to reduce the
potential for mucosal exposure to the vector. In most such research, these levels of
containment are also expected to be appropriate even when producing large volumes of
HIV-1 vectors (>10 L).
The appropriate containment level for specific lentivirus vector research is, of course,
determined following a complete risk assessment and local IBC review. The following
sections discuss some considerations which should form an important part of the
biosafety assessment for research involving lentivirus vectors.
Potential for generation of replication competent lentivirus (RCL) from HIV1 based lentivirus vectors: The potential for generation of RCL from HIV-1 based
lentivirus vectors depends upon several parameters, the most important of which are:
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The number of recombination events necessary to reassemble a replication
competent virus genome, and
The number of essential genes that have been deleted from the
vector/packaging system.
On this basis, later generation lentivirus vector systems are likely to provide for a greater
margin of personal and public safety than earlier vectors, because:
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They use a heterologous coat protein (e.g., VSV-G) in place of the native
HIV-1 envelope protein (However, the use of the certain coat proteins, such
as VSV-G, may broaden the host cell and tissue tropism of lentivirus vectors,
which should also be considered in the overall safety assessment by the
IBC),
They separate vector and packaging functions onto four or more plasmids,
and
They include additional safety features (e.g., they do not encode Tat, which is
essential for replication of wild-type HIV-1).
In contrast, earlier vector systems (such as two-plasmid vector systems) may have a
higher potential for generation of RCL.
RCL testing: The National Gene Vector Laboratory (NGVL) has produced over 60
liters of HIV-1 vector and has screened supernatant and cells from different vector
systems, using different assays, without detecting RCL (K. Cornetta, personal
communication of unpublished data). This suggests that the frequency of RCL
generation using lentivirus vectors is very low. It may not, however, be zero. There is a
need for continued investigation of RCL generation using lentivirus vectors, in order to
inform and advance the field of lentivirus vector technology.
The FDA requires that lentiviral vector stocks used in human clinical trials be tested for
RCL. Individual research laboratories conducting preclinical research often use only
small volumes (e.g., a few milliters) of lentivirus vectors expressing lower risk transgenes
such as GFP. While these laboratories are not mandated to characterize vector stocks,
such testing should be encouraged. However, RCL testing requires expertise with the
appropriate assays and such expertise may not be available in laboratories that do not
work regularly with infectious lentiviruses. In such laboratories, the use of a positive
control may increase risk to the investigator as compared to use of the test material.
IBCs may make containment assignments without requiring such testing by undertaking
a risk assessment that considers the nature of the specific vector system being used and
overall past experience with the system.
Animal studies: Some animals, such as wild-type mice, cannot support replication of
infectious HIV-1. As a result, the potential for shedding of RCL from such animals is very
low (even if RCL were present in the original vector inoculum). IBCs may consider the
biosafety issues associated with animal husbandry and housing after the initial injection
separately from the initial inoculation itself. In general, the initial delivery of vector should
be performed under BL2 or BL2-N, according to the animal model, or under enhanced
BL-2/BL2-N containment (see "General containment considerations"), so as to minimize
the risk of autoinoculation by the investigator. However, it may be permissible to reduce
the containment level at some point following vector delivery. For example, if there is no
expectation of infection (see below), the site of inoculation has been thoroughly
cleansed, and the bedding changed, it may be acceptable to consider reducing
containment from BL2/BL2-N to BL1/BL1-N within a few days (the specific time period
can be specified by the local IBC, and may vary anywhere from 1-7 days depending on
local and experimental considerations). Animals engrafted with human cells or animal
hosts that are permissive for HIV-1 replication constitute a special case, in light of their
potential to support replication of infectious HIV-1. Use of lentivirus vectors in these
animals requires a higher level of containment.
Other lentivirus vectors: Some non-human lentivirus vectors (e.g., FIV, SIV, EIAV,
etc.) are also in use. Of these, the most frequently encountered are feline
immunodeficiency virus (FIV) vectors. In the Appendix B-V of the NIH Guidelines, a
containment level appropriate for Risk Group 1 agents is recommended for use of
certain animal viral etiologic agents not associated with disease in healthy human adults.
However, replication-defective vectors in which a heterologous envelope (such as VSVG) is used for vector packaging may require BL2 containment in the laboratory setting,
since these vectors have the potential to transduce human cells, and thus have the
potential to cause insertional mutagenesis. Under circumstances in which mice are not
permissive hosts for FIV replication, BL1 containment may be acceptable for mouse
housing and husbandry when dealing with mice that have received FIV vectors (subject
to the considerations noted above).
Summary: A comprehensive risk assessment and determination of containment for
research with lentiviral vectors should consider the nature of the vector system,
transgene insert, and type of manipulations involved. For many experiments, either BL-2
or enhanced BL-2 will be appropriate. Examples of biosafety considerations and risk
assessments for three different scenarios are included below.
Examples of Biosafety Considerations
Vector Considerations:

Potential for generation of RCL
o Vector and packaging functions separated onto multiple plasmids
o Deletion of viral genes
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Viral Env used in packaging system
o Non-native Env (decrease potential for generation of RCL)
o Coat protein that increases species or cell type tropism of parent
virus (e.g., VSV-G)
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Safety modifications (e.g., no expression of Tat)
Transgene Considerations:
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Oncogene
Non-oncogene
Vector Generation Considerations:
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Laboratory scale
Large scale
Animal Research Considerations:
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Permissive host
Non-permissive host
Animal engrafted with permissive cells
Vector Administration (e.g., injection)
Housing and husbandry
Practices, Containment Equipment and Training Considerations:
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Training in use of PPE
Availability of safety equipment (e.g., sealed centrifuge rotor cups)
Laboratory-specific safety and spill cleanup protocols
Availability of on-site occupational health support in the event of accident
Biosafety Considerations and Risk Levels
Biosafety
Considerations
Higher Risk
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Vector packaging functions
on two plasmids
Expression of viral genes
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Vector and packaging
functions separated onto
multiple plasmids
Deletion of viral genes
Transgene
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Oncogene
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Non-oncogene
Vector
Generation
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Large scale
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Laboratory scale
Animal Hosts
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Permissive host
Animals engrafted with
human cells
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Non-permissive host
Animal
Manipulation
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Vector administration (e.g.,
use of sharps during
injection
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Housing and husbandry (no
use of sharps)
Vector Design
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Lower Risk
EXAMPLE SCENARIOS
EXAMPLE ONE: In vitro study A: Use of a 4-plasmid derived lentivirus
vector encoding siRNA against Lck in primary human T cells.
Considerations
1. What is the amount of vector to be produced? A = LOW (100 ml)
2. What is the nature of the vector? A = 4-Plasmid System
3. What is the nature of the insert? A = Non-Oncogenic
Tentative Safety Assessment = BL2
(Note that the use of primary human cells would require BL2 containment,
independent of the vector, as well as use of Universal Precautions and
compliance with the OSHA standard for Bloodborne Pathogens -- 29 CFR
1910.1030)
EXAMPLE TWO: In vitro study B: Use of a 2-plasmid derived lentivirus
vector encoding luciferase in a human cell line (A549 cells).
Considerations
1. What is the amount of vector to be produced? A = LOW (100 ml)
2. What is the nature of the vector? A = 2-Plasmid System (non-commercial)
3. What is the nature of the insert? A = Non-Oncogenic
Tentative Safety Assessment = BL2 enhanced
BL2 "enhanced" stipulations might include:
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Avoidance of needles and sharps, where possible
Use of a containment hood for all work with the vector (including the
loading and unloading of centrifuge rotors, which should have an aerosoltight seal)
Use of personal protective equipment [PPE] designed to prevent a
mucosal exposure/splash to the face and exposure of hands (especially in
persons with broken skin or open cuts)
A requirement for an in-person consultation between biosafety staff and
lab personnel prior to initiation of experiments
EXAMPLE THREE: In vivo study A: Use of a 4-plasmid derived lentivirus
vector encoding brain-derived neurotrophic factor (BDNF) in mouse brain.
Considerations
1. What is the amount of vector to be produced? A = LOW (100 ml)
2. What is the nature of the vector? A = 4-Plasmid System
3. What is the nature of the insert? A = Non-Oncogenic (*: see below)
4. What is the nature of the animal host? A = Non-permissive for HIV-1
Tentative Safety Assessment = BL2 for lab work and initial injection of mice
(which would probably be done using a stereotactic frame); after 1-7 days,
animals could be moved to BL1 containment.
Added explanation:
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Even though BDNF is a growth factor for neurons, it has no known
oncogenic activity for skin or blood cells that might be the target of a
potential needle stick. Hence, this insert would not automatically trigger a
requirement for increased biocontainment.
Stereotactic injection frames cannot easily be placed into a laminar flow
hood, and may use a syringe or pulled glass pipette for inoculation; they
may also use a pump to ensure a slow rate of delivery of the agent. BL2
containment does NOT require the use of a biosafety cabinet, and is
therefore compatible with the use of a stereotactic frame, even if that
frame is not contained within a laminar flow cabinet.
Additional points to consider:
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An in-person consultation between biosafety staff and lab personnel prior
to initiation of experiments may be a useful stipulation
One might also impose additional biosafety enhancements during the
injection process, perhaps by requiring use of additional PPE above and
beyond the stipulated requirements associated with BL2/BL2-N. See
Example 2 for examples of such stipulations.
APPENDIX
Sections from the NIH Guidelines
General Considerations
Section II-A. Risk Assessment
Section II-A-3. Comprehensive Risk Assessment. BL2 containment is recommended
for activities involving all blood-contaminated clinical specimens, body fluids, and tissues
from all humans, or from HIV – or HBV-infected or inoculated laboratory animals.
Activities such as the production of research-laboratory scale quantities of HIV or other
bloodborne pathogens, manipulating concentrated virus preparations, or conducting
procedures that may produce droplets or aerosols, are performed in a BL2 facility using
the additional practices and containment equipment recommended for BL3. Activities
involving industrial scale volumes or preparations of concentrated HIV are conducted in
a BL3 facility, or BL3 Large Scale if appropriate, using BL3 practices and containment
equipment.
Appendix
Section III-D. Experiments That Require Institutional Biosafety Committee
Approval Before Initiation
Section III-D-3. Experiments Involving the Use of Infectious DNA or RNA Viruses
or Defective DNA or RNA Viruses in the Presence of Helper Virus in Tissue Culture
Systems. Recombinant DNA or RNA molecules derived therefrom, which contain less
than two-thirds of the genome of any eukaryotic virus (all viruses from a single Family
(see Section V-J, Footnotes and References of Sections I-IV) being considered identical
(see Section V-K, Footnotes and References of Sections I-IV), are considered defective
and may be used in the absence of helper under the conditions specified in Section III-E1, Experiments Involving the Formation of Recombinant DNA Molecules Containing No
More than Two-Thirds of the Genome of any Eukaryotic Virus.
Section III-E. Experiments That Require Institutional Biosafety Committee Notice
Simultaneous with Initiation
Section III-E-1. Experiments Involving the Formation of Recombinant DNA
Molecules Containing No More than Two-Thirds of the Genome of any Eukaryotic
Virus. Recombinant DNA molecules containing no more than two-thirds of the genome
of any eukaryotic virus (all viruses from a single Family being considered identical [see
Section V-J, Footnotes and References of Sections I-IV]) may be propagated and
maintained in cells in tissue culture using BL1 containment. For such experiments, it
must be demonstrated that the cells lack helper virus for the specific Families of
defective viruses being used. If helper virus is present, procedures specified under
Section III-D-3, Experiments Involving the Use of Infectious Animal or Plant DNA or RNA
Viruses or Defective Animal or Plant DNA or RNA Viruses in the Presence of Helper
Virus in Tissue Culture Systems, should be used. The DNA may contain fragments of
the genome of viruses from more than one Family but each fragment shall be less than
two-thirds of a genome.
Animal Studies
Section III-D-4-a. Recombinant DNA, or DNA or RNA molecules derived therefrom, from
any source except for greater than two-thirds of eukaryotic viral genome may be
transferred to any non-human vertebrate or any invertebrate organism and propagated
under conditions of physical containment comparable to BL1 or BL1-N and appropriate
to the organism under study (see Section V-B, Footnotes and References of Sections IIV). Animals that contain sequences from viral vectors, which do not lead to
transmissible infection either directly or indirectly as a result of complementation or
recombination in animals, may be propagated under conditions of physical containment
comparable to BL1 or BL1-N and appropriate to the organism under study. Experiments
involving the introduction of other sequences from eukaryotic viral genomes into animals
are covered under Section III-D-4-b, Experiments Involving Whole Animals. For
experiments involving recombinant DNA-modified Risk Groups 2, 3, 4, or restricted
organisms, see Sections V-A, V-G, and V-L, Footnotes and References of Sections I-IV.
It is important that the investigator demonstrate that the fraction of the viral genome
being utilized does not lead to productive infection. A U.S. Department of Agriculture
permit is required for work with plant or animal pathogens (see Section V-G, Footnotes
and References of Sections I-IV).
APPENDIX B. CLASSIFICATION OF HUMAN ETIOLOGIC AGENTS ON THE BASIS
OF HAZARD
Appendix B-III-D. Risk Group 3 (RG3) - Viruses and Prions
Retroviruses
--Human immunodeficiency virus (HIV) types 1 and 2
BL2 Facilities
Appendix G-II-B-3. Containment Equipment (BL2)
Appendix G-II-B-3-a. Biological safety cabinets (Class I or II) (see Appendix G-III-L,
Footnotes and References of Appendix G) or other appropriate personal protective or
physical containment devices are used whenever:
Appendix G-II-B-3-a-(1). Procedures with a high potential for creating aerosols are
conducted (see Appendix G-III-O, Footnotes and References of Appendix G). These
may include centrifuging, grinding, blending, vigorous shaking or mixing, sonic
disruption, opening containers of materials whose internal pressures may be different
from ambient pressures, intranasal inoculation of animals, and harvesting infected
tissues from animals or eggs.
Appendix G-II-B-3-a-(2). High concentrations or large volumes of organisms containing
recombinant DNA molecules are used. Such materials may be centrifuged in the open
laboratory if sealed beads or centrifuge safety cups are used and if they are opened only
in a biological safety cabinet.
Appendix G-II-B-4. Laboratory Facilities (BL2)
Appendix G-II-B-4-a. The laboratory is designed so that it can be easily cleaned.
Appendix G-II-B-4-b. Bench tops are impervious to water and resistant to acids, alkalis,
organic solvents, and moderate heat.
Appendix G-II-B-4-c. Laboratory furniture is sturdy and spaces between benches,
cabinets, and equipment are accessible for cleaning.
Appendix G-II-B-4-d. Each laboratory contains a sink for hand washing.
Appendix G-II-B-4-e. If the laboratory has windows that open, they are fitted with fly
screens.
Appendix G-II-B-4-f. An autoclave for decontaminating laboratory wastes is available.